Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation

Authors

  • Ali Hayek,

    1. Institute of Physics and Chemistry of Materials of Strasbourg, Organic Materials Group, CNRS and Université Louis Pasteur-Strasbourg I (UMR 7504), 23 rue du Loess, 67034 Strasbourg, France
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  • Alexei Grichine,

    1. University Joseph Fourier-Grenoble I, Institute Albert Bonniot – IFR 73, Platform “Optical microscopy–cell imaging,” BP 170, La Tronche, 38042 Grenoble Cedex 9, France
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  • Thomas Huault,

    1. Laboratoire de Spectrométrie Physique, CNRS and Université Joseph Fourier-Grenoble I (UMR 5588), BP 87, 38402 Saint Martin d'Hères, France
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  • Clément Ricard,

    1. INSERM U594 “Functional and Metabolic Neuroimaging,” Academic Hospital of Grenoble, Pavillon B, BP 217, 38043 Grenoble, France
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  • Frédéric Bolze,

    1. Institute of Physics and Chemistry of Materials of Strasbourg, Organic Materials Group, CNRS and Université Louis Pasteur-Strasbourg I (UMR 7504), 23 rue du Loess, 67034 Strasbourg, France
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  • Boudewijn Van Der Sanden,

    1. INSERM U647 “Medical Applications of Synchrotron Radiation”, European Synchrotron Radiation Facility, 6 rue Jules Horowitz, 38043 Grenoble, France
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  • Jean-Claude Vial,

    1. Laboratoire de Spectrométrie Physique, CNRS and Université Joseph Fourier-Grenoble I (UMR 5588), BP 87, 38402 Saint Martin d'Hères, France
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  • Yves Mély,

    1. Institut Gilbert Laustriat, CNRS (UMR 7175), Faculté de Pharmacie, 74, route du Rhin, BP 60024, 67401 Illkirch-Graffenstaden, France
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  • Alain Duperray,

    1. INSERM U578, 38706 Grenoble, France
    2. Université Joseph Fourier-Grenoble I, Groupe de Recherche sur le Cancer du Poumon, Institut Albert Bonniot, (IFR 73), La Tronche 38706 Grenoble, France
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  • Patrice Lilian Baldeck,

    1. Laboratoire de Spectrométrie Physique, CNRS and Université Joseph Fourier-Grenoble I (UMR 5588), BP 87, 38402 Saint Martin d'Hères, France
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  • Jean-François Nicoud

    Corresponding author
    1. Institute of Physics and Chemistry of Materials of Strasbourg, Organic Materials Group, CNRS and Université Louis Pasteur-Strasbourg I (UMR 7504), 23 rue du Loess, 67034 Strasbourg, France
    • Institute of Physics and Chemistry of Materials of Strasbourg, Organic Materials Group, CNRS and Université Louis Pasteur-Strasbourg I (UMR 7504), 23 rue du Loess, 67034 Strasbourg, France
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Abstract

Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood–brain barriers. They are characterized by two-photon absorption cross-sections of 100–150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.

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